Small Molecule Inhibitors of Botulinum Neurotoxins

ABSTRACT

Disclosed herein are methods of inhibiting the activity of Botulinum neurotoxin A metalloprotease with the compounds disclosed herein. Also disclosed are methods of treating, inhibiting or preventing intoxication caused by bacteria of at least one bacterial strain in a subject, and pharmaceutical and cosmetic compositions comprising the compounds disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 60/707,531, filed 12 Aug. 2005, and 60/723,442,filed 5 Oct. 2005, both of which are herein incorporated by reference intheir entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made by employees of the United States Army MedicalResearch and Materiel Command, which is an agency of the United StatesGovernment. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds which inhibit botulinumneurotoxin. In particular, the present invention relates to compoundswhich inhibit botulinum neurotoxin serotype A light chain (BoNT/A LC)metalloprotease activity.

2. Description of the Related Art

Botulinum neurotoxins (BoNTs) are produced by spore forming anaerobicbacteria Clostridium botulinum, and are among the most lethal ofbiological poisons (LD₅₀=0.001 μg per Kg). See Schmidt & Stafford (2003)Appl. Environ. Microbiol. 69:297-303; Kessler & Benecke (1997)Neurotoxicology 18:761-770; and Burnett et al. (2005) Nat Rev DrugDiscov 4(4):281-297. Seven immunologically distinct BoNT serotypes(designated A-G) have been identified. See Simpson, L. L. (1989)BOTULINUM NEUROTOXIN AND TETANUS TOXIN, Academic Press, New York.

Exposure to BoNTs, for example, through contaminated food, can result inlife threatening flaccid paralysis. See Shapiro, et al. (1998) Ann.Intern. Med. 129:221-228. Furthermore, BoNTs have been weaponized inhighly toxic aerosol form, and consequently pose a significant threat toboth to civilian and military populations. See Franz, et al. (1997) JAMA278:399-411; and Amon, et al. (2001) JAMA 285:1059-1070.

As indicated, these enzymes have been weaponized in aerosol medium, andairborne release or direct contamination (e.g. foodstuffs) representsignificant threats to both military and civilian populations. SeePaddle, B M (2003) J Appl Toxicol 23(3):139-170; Clarke, S C (2005) Br JBiomed Sci 62(1):40-46; Hicks et al. (2005) Curr Med Chem 12(6):667-690;and Josko, D (2004) Clin Lab Sci 17(1):30-34. And, with the increaseduse of BoNTs as therapies for a range of medical conditions andsuperficial cosmetic purposes, there is the increased potential foraccidental overdosing. Furthermore, as the popularity of BoNTs astherapeutics continues to grow, these enzymes are increasingly beingmanufactures overseas, where less strict controls may allow clandestineorganizations to obtain large quantities of these toxins in veryconcentrated, pure, and easily stored formulations. See Comella &Pullman (2004) Muscle Nerve 29(5):628-644; Gormley et al. (1997) MuscleNerve Suppl 6:S14-20; Marks, J D (2004) Anesthesiol Clin North America22(3):509-532, vii; Noonan & Adler (2002) Newsweek 139(19):50-56, 58;O'Brien, C F (2001) Adv Neurol 87:265-269; O'Brien, C F (2002) Clin JPain 18(6 Suppl):S182-190; O'Brien, D (2003) J Perianesth Nurs18(2):126-134; Rossetto (2001) Toxicon 39(1):27-41; Shukla & Sharma(2005) Crit. Rev Microbiol 31(1):11-18; and Turton et al. (2002) TrendsBiochem Sci 27(11):552-558. As a result, there is an urgent need fortherapeutic countermeasures against BoNTs. See Goodnough, et al. (2002)FEBS Lett. 513:163-168.

BoNT is secreted as a holotoxin composed of two peptide chains that arelinked by a disulfide bridge. See Lacy & Stevens (1999) J. Mol. Biol.291:1091-1104. The heavy chain is responsible for: (1) targeting andbinding to surface receptors on nerve terminals; (2) translocation intothe neuronal cytosol via the formation of a low pH endosome; and (3)protecting the substrate binding cleft of the light chain prior toneuronal internalization. See Turton, et al. (2002) Trends Biochem. Sci.27:552-558; and Singh, B. R. (2000) Nat. Struct. Biol. 7 (2000) 617-619.The light chain, which dissociates from the heavy chain in the lowendosomal pH, is released into the cytosol where it acts as a zincmetalloprotease that cleaves soluble NSF-attachment protein receptor(SNARE) proteins: synaptosomal-associated protein of 25 kDa (SNAP-25),synaptobrevin, and syntaxin. BoNT serotypes A, C, and E cleave SNAP-25;serotypes B, D, F, and G cleave synaptobrevin; and serotype C can alsouse syntaxin as substrate. See Binz, et al. (1994) J. Biol. Chem.269:1617-1620; Schiavo, et al. (1992) Nature 359:832-835; Schiavo, etal. (1993a) J. Biol. Chem. 268:23784-23787; Schiavo, et al. (1993c) J.Biol. Chem. 268:11516-1151915; Schiavo, et al. (1993b) J. Biol. Chem.269:20213-20216; and Blasi, et al. (1993b) EMBO J. 12:4821-4828. Withoutfunctional SNARE complexes, acetylcholine is not released intoneuromuscular junctions, thereby leading to paralysis.

Research to identify peptide and small molecule inhibitors of BoNTserotype A (BoNT/A) has targeted both holotoxin translocation and lightchain (BoNT/A LC) metalloprotease activity. Sheridan et al. andDeshpande et al. have shown that a number of antimalarial agentsinterfere with BoNT/A translocation into nerve cytoplasm. See Sheridan,et al. (1997) Toxicon 35:1439-1451; and Deshpande, et al. (1997) Toxicon35:433-445.

Specifically, it has been shown that several antimalarial compounds actsubsequent to toxin binding to cell-surface receptors, and it has beenhypothesized that these agents inhibit BoNT/A cytosol entry by raisingendosomal pH (an endosomal pH of 5.5 or lower is needed for release intothe cytoplasm). Hayden et al. have found that BoNT/A LC is inhibited bymM concentrations of known protease inhibitors: captopril, lysinopril,and enalapril. See Hayden, et al. (2003) J. Appl. Toxicol. 23:1-7. Inthe same study, it was also reported that a number of short peptides,from specific “hinge” libraries, inhibit BoNT/A LC activity by as muchas 51% at concentrations as low as 0.5 μM. Using a chromatographicmethod, Schmidt et al. identified the peptide motif CRATKML as a potentinhibitor. See Schmidt, et al. (1998) FEBS Lett. 435:61-64. In asubsequent study, the Cys residue of CRATKML was replaced with thiolcontaining organic moieties, and it was found that a2-mercapto-3-phenylpropionly containing derivative was the mosteffective (Ki=0.3 μM). See Schmidt & Stafford (2002) FEBS Lett.532:423-426.

Neither the currently available BoNT antitoxin nor antibodies cancounter these toxins once they are inside neurons; currently, criticalcare mechanical ventilation is the only treatment option. However, theeffects of internalized BoNTs can last for months, and mechanicalventilation would be impractical if even a limited number of individualswere simultaneously intoxinated. See Meunier et al. (2003) Mol CellNeurosci 22(4):454-466; and Eleopra et al. (1998) Neurosci Lett256(3):135-138. Furthermore, antitoxin administration would precludevaccinated individuals from any form of highly beneficial BoNT medicaltherapy.

Thus, a need exists for small molecule (non-peptidic) inhibitors ofBoNT/A LC metalloprotease activity.

SUMMARY OF THE INVENTION

The present invention generally relates to compounds that inhibit BoNT/ALC metalloprotease activity.

In some embodiments, the present invention provides a method ofinhibiting the activity of the Botulinum neurotoxin A metalloproteasewhich comprises contacting Botulinum neurotoxin A metalloprotease withat least one compound having the following structural formula:

n is 1 or 2;

X¹, X², X³, X⁴, X⁵ and X⁶ are each independently N, S, O, SO₂, CR⁷ orNR⁸ and at least one of X¹ or X² is N, S, O, SO₂, or NR⁸;

L is a linker which may be a direct bond or

where Z is an optionally substituted alkyl, alkenyl, dialkenyl,trialkenyl, or aryl, or C(O)NH; and

R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are each independently hydrogen, amino,amine with stabilized carbocations, carboxyl, optionally substitutedalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,alkoxy, aryoxy, cycloalkoxy, heteroaryloxy, alkoxycarbonyl, alkylamino,carbamoyl, alkylaminocarbonyl, alkylsulfhydryl, alkylhydroxymate; and

R⁸ is hydrogen, OH, a halogen, or an optionally substituted alkyl.

In some embodiments, at least one of R′, R², R³, or R⁴ is hydrogen,amidine, 2-imidazoline, amino, guanidine, methyl,aminomethyl-hydroxamine, or methylamine-guanidine. In some embodiments,R⁵ is hydrogen, amidine, 2-imidazoline, amino, guanidine, methyl,aminomethyl-hydroxamine, methylamine-guanidine, 4-oxy-benzamidine,1H-indole-6-caboxamidine, or 1H-indole-5-carboxamidine. In someembodiments, R⁶ is hydrogen, amidine, benzamidine, benzimidazoline,imidazoline, guanidine, imidazole, oxazole, benzofuran-2-yl-imidazoline,benzofuran-2-yl-amidine, benzofuran-2-yl-guanidine,benzothiophene-2-yl-imidazoline, benzothiophene-2-yl-amidine,benzene-2-yl-amidine, benzofuran-2-yl-imidazole, orbenzofuran-2-yl-oxazole. In some embodiments, at least one of X¹ or X²is N, NH, S, O, SO₂, CH, C—CH₃, C-phenyl, N-ethanol, N-chloroethyl,C-amino, C-(2-indole-6-imidazoline), C-(2-indole-6-amidine),C-(2-indole-5-imidazoline), or C-(2-indole-5-amidine). In someembodiments, at least one of X³, X⁴, X⁵, or X⁶ is N, NH, S, O, SO₂, orCH. In some embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷is —H, —CH₃, —NH₂,

In some embodiments, R⁵ is ˜NH₂,

In some embodiments, R⁶ is

In some embodiments, R⁷ is —H, —CH₃, —NH₂,

In some embodiments, R⁸ is —H, —(CH₂)₂OH, or —(CH₂)₂Cl. In someembodiments, L is a direct bond,

In some embodiments, the compound has the following structural formulae:

In some embodiments, the compound is NSC 92833, NSC 103699, NSC 103701,NSC 130681, NSC 240890, NSC 240891, NSC 240893, NSC 240894, NSC 240895,NSC 240896, NSC 240897, NSC 240898, NSC 240899, NSC 240900, NSC 266472,NSC 266474, NSC 266475, NSC 266476, NSC 266477, NSC 266482, NSC 278995,NSC 278996, NSC 278997, NSC 278999, NSC 290107, NSC 290108, NSC 290109,NSC 290111, NSC 291103, NSC 294199, NSC 294200, NSC 294201, NSC 294202,NSC 294203, NSC 294204, NSC 294206, NSC 294207, NSC 294208, NSC 294494,NSC 300509, NSC 300510, NSC 300511, NSC 300512, NSC 302569, NSC 308569,NSC 308570, NSC 308571, NSC 308572, NSC 308573, NSC 308574, NSC 317880,NSC 317881, NSC 317883, NSC 317884, NSC 317885, NSC 317886, NSC 317887,NSC 328398, NSC 330687, NSC 330688, NSC 330689, NSC 330690, NSC 341082,NSC 341907, NSC 341909, NSC 341910, NSC 341911, NSC 352341, NSC 369718,NSC 369721, NSC 607617, or NSC 12155. In some embodiments, the compoundis NSC 341909, NSC 308574, NSC 240898, NSC 341907, NSC 266472, NSC330690, NSC 278999, NSC 308571, NSC 290107, NSC 290108, NSC 294200, NSC317884, NSC 317884, NSC 294203, NSC 294494, NSC 317881, NSC 330688, NSC317886, NSC 317833, NSC 328398 NSC 352341, NSC 294204, NSC 341911, NSC300511, NSC 607617, NSC 294202, NSC 317880, NSC 240899, NSC 294201, NSC291103, NSC 308573, NSC 290109, NSC 294206, NSC 308570, NSC 294199, NSC369723, or NSC 300510.

In some embodiments, the present invention provides a compound havingthe following structural formula:

wherein R^(a) and R^(b) are each independently —CN, —CONH₂, or—C(═NH)NH₂; X is —NH or O; and Y is N or —CH,

In some embodiments, the present invention provides a method ofinhibiting the activity of Botulinum neurotoxin A metalloprotease whichcomprises contacting Botulinum neurotoxin A metalloprotease with atleast one compound provided herein.

In some embodiments, the present invention provides a method oftreating, inhibiting or preventing a subject from being intoxicated byBotulinum toxin which comprises administering to the subject atherapeutically effective amount of at least one compound providedherein.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising at least one compound provided herein and apharmaceutically acceptable carrier.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are intended toprovide further explanation of the invention as claimed. Theaccompanying drawings are included to provide a further understanding ofthe invention and are incorporated in and constitute part of thisspecification, illustrate several embodiments of the invention, andtogether with the description serve to explain the principles of theinvention.

DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIG. 1 is a table which exemplifies some compounds of the presentinvention, their K_(i) values and pharacophore query fits and distancesbetween pharmacophore components.

FIG. 2A shows that NSC 240898 lacks substantial cytotoxic effects onchick spinal motor neurons at concentrations up to about 40 μM. Stainingfor tubulin (green), actin filaments (red), and DNA (blue) show no grossmorphological abnormalities in neurons after 3.5 hours incubation witheither 10 nM BoNT/A holotoxin or inhibitor+10 nM BoNT/A holotoxin whencompared to untreated neurons. Scale bar=20 μm.

FIG. 2B is a Western blot revealing a dose-dependent NSC 240898inhibition of BoNT/A SNAP-25 cleavage.

FIG. 2C provides the densitometric scans of the bands in the Westernblot of FIG. 2B graphically as a ratio of cleaved to intact SNAP-25 forthe concentrations of NSC 240898.

FIGS. 3A-3B are graphs of the isothermal titration calorimetry (ITC) ofthe NSC 240848: BoNT/A LC interaction.

FIG. 3A shows raw data obtained for 30 injections of 10 μl of 1.3 mM NSC240848 solution into the sample cell containing 50 μM BoNT/A LC (aftersubstraction of the integration baseline).

FIG. 3B shows normalized integrated enthalpies plotted against the molarratio NSC 240898: BoNT/A LC. The solid line corresponds to the best fitcurve obtained by non-linear least square fit minimization. The bindingfollowed a 1:1 stoichiometry and is largely entropy-driven. Due to thelow affinity of the interaction, a large excess of the inhibitor wasnecessary to drive the titration to saturation. Protein concentrationwas determined by amino acid analysis (Molecular Structure Facility,Univ. Calif Davis), and NSC 240898 concentration was confirmed by UV/Visabsorbance measurements.

DETAILED DESCRIPTION OF THE INVENTION

The pharmacophore model for BoNT/A LC metalloprotease inhibitionprovided in U.S. Patent Publication No. 20050153945, which is hereinincorporated by reference, was used to screen for small moleculecompound candidates that would likely inhibit BoNT/A LC metalloproteaseactivity. As disclosed herein, several imidazoline compounds wereidentified and found to inhibit BoNT/A LC metalloprotease activity.

Identification of Candidate Small Molecule Inhibitors

Candidate small molecule inhibitors of BoNT/A LC metalloproteaseactivity were identified by pharmacophore analysis, molecular dynamicand molecular docketing studies.

1. Initial BoNT/A LC Pharmacophore

Previously, Burnett et al. identified a diverse range of compounds thatinhibit the metalloprotease activity of the BoNT/A LC. See U.S. patentPublication No. US 20050153945 and Burnett et al. (2003) Biochem.Biophys. Res. Commun. 310 (1):84-93, which are herein incorporated byreference. A high-throughput fluorescence-based assay was initially usedto screen the NCI diversity set, a collection of 1990 molecules thatwere selected to cover a wide range of conformational space, and at thesame time provide pharmacophore diversity and structural rigidity. Thena HPLC-based assay known in the art was used to eliminate falsepositives. See Schmidt et al. (2003) Appl Environ Microbiol 69:297-303,which is herein incorporated by reference. The resulting set ofinhibitors, which were tested at 20 μM concentrations, in the presenceof 0.1 mM substrate, comprised 21 structurally diverse compounds withpotencies ranging from about 14% to about 100% inhibition of BoNT/A LCmetalloprotease activity. The compounds possessing greater than about40% inhibition were NSC 625324 (silver sulfadiazine), NSC 661755(Michellamine B), NSC 357756, NSC 119889, NSC 86372, NSC 130796 and NSC402959. Congeneric series ofN,N-bis(7-chloroquinolin-4-yl)alkanediamines andN,N-bis(7-chloroquinolin-4-yl)heteroalkanediamines (collectivelyreferred to as bisquinolines or BQs) were also examined for BoNT/A LCinhibition. See Vennerstrom et al. (1992) J Med Chem 35:2129-2134; andVennerstrom et al. (1998) J Med Chem 41:4360-4364, which are hereinincorporated by reference. These compounds were found to be non-zincchelators. Five readily available antimalarial drugs—amodiaquine,chloroquine, quinacrine, quinidine, and quinine—were also tested, asthey share similar structural features with the BQs. The antimalarialdrugs that were found to inhibit BoNT/A LC protease activity in ourstudy have previously been shown to increase the time to BoNT/Aholotoxin induced muscle paralysis by interfering with toxintranslocation into the nerve cytoplasm. See Deshpande et al. (1997)Toxicon 35:433-445; and Sheridan et al. (1997) Toxicon 35:1439-1451,which are herein incorporated by reference. Thus, as provided herein,the compounds of the present invention may be used to inhibit bothBoNT/A entry into the cytoplasm and protease activity of the LC.

2. BoNT/A LC Molecular Dynamics

X-ray crystal structures of BoNT/A have been solved; however, they leavean information gap with regard to the periodic motion of the LC. Inparticular, conformational changes in and around the substrate bindingcleft could have profound effects on the design of inhibitors.Consequently, a molecular dynamics study was conducted to explore themotion of the BoNT/A LC over time. See Burnett et al. (2005) Bioorg MedChem 13:333-341, which is herein incorporated by reference. Results fromthese analyses indicated that LC α-helices and 13-sheets remainrelatively unchanged over the course of a 1 ns dynamics trajectory.However, significant conformational flexibility in surface loopsbordering the substrate binding cleft was observed. Extensive analysesindicated that these loops might possess the ability to partially shieldthe substrate binding cleft from the solvent front. Based on moleculardocking studies with identified inhibitors, the observed reorientationof these loops toward the enzyme's substrate binding cleft may serve toprovide additional residue:inhibitor (or substrate) contacts, andfacilitate inhibitor desolvation. This information was used to refinethe pharmacophore for BoNT/A LC inhibition.

3. Molecular Docking of mpp-RATKML.

To improve the structure-based understanding of BoNT/A LC, the bindingmode for a potent inhibitor, 2-mercapto-3-phenylpropionyl-RATKML (SEQ IDNO:1) (mpp-RATKML₁=330 nM), was studied in order to reveal steric spaceand residue contacts not ascertained by the original pharmacophore modeland molecular dynamic studies.

To ensure that we generated the most realistic model possible, weadopted a number of strict molecular docking criteria: (1) theinhibitor's sulfur (S), which coordinates the enzyme's catalytic Zinc(Zn), must do so with a distance and incidence angle consistent withavailable data from the Cambridge Structural Database (CSD) and theProtein Data Bank (PDB); (2) the binding mode must adhere to thehydrophobicity-first rule of molecular docking; 3) the inhibitor'sintermolecular non-bonded contacts and its intramolecular conformationmust be hydropathically and structurally feasible; and 4) the bindingmode of the inhibitor must rationalize its structure activityrelationship (SAR). See Schmidt & Stafford (2002) FEBS Lett 532:423-426;Schmidt et al. (1998) FEBS Lett 435:61-64; Alberts et al. (1998) ProteinSci. 7:1700-1716; and Roe et al. (1999) J. Mol. Model. 5:134-140, whichare herein incorporated by reference.

An acceptable binding mode for the pseudo-peptide using an energyrefined X-ray crystal structure of the BoNT/A LC possessing the highestresolution (PDB 1E1H, resolution=1.8 Å) was not readily identified.When, however, an enzyme conformer from a previous dynamics trajectoryof 1E1H was used, a biochemically feasible binding mode was obtained. Inbrief, for the mpp portion of the inhibitor, the phenyl substituentengaged in face-to-face π stacking with the side chain phenyl of Phe 193(in addition to maintaining favorable hydrophobic contacts with the Phe162 and Thr 219 side chains). Furthermore, the S—Zn interaction wasmaintained within a specified distance and an angle of incidence thatwere verified by examining numerous structures form the CSD and PDB. Theside chain methylenes of the inhibitors Arg packed with goodhydrophobic-hydrophobic complementarity in the hydrophobic pocketcreated by loop 1 (residues 48-78) reorientation during the dynamicssimulation. Desolvation of the hydrophobic portion of the Arg side chaindictated the position of its cationic guanidinium, which engaged inhydrogen bonds with the side chain carboxylates of residues Glu 63 (ofloop 1) and Glu 163 (of the polar contact region).

The inhibitor's Ala, Thr, Lys, Met and Leu residues were systematicallydocked and optimized in the substrate binding cleft using the samestrict docking criteria indicated above. The Ala residue is buried inthe substrate binding cleft, resting behind the side chain imidazole ofH is 226, near the side chain methylene of Cys 164, and away from theside chain carboxylate of Glu 261. The amphipathic Thr side chain ispositioned so that its methyl substituent points toward the substratebinding cleft and engages in favorable hydrophobic contacts with the Val67 side chain, while its polar hydroxyl moiety is oriented toward thesolvent. The side chain methylenes of the inhibitor's Lys residuedesolvate by lining up parallel to the side-chain methylenes of Lys165—this positions the residue's cationic Nζ within hydrogen bondingdistance to both the side chain carboxylate of Glu 54 and the side chainamide carbonyl oxygen of Asn 52 (both of these residues are brought intocloser association with the BoNT/A LC substrate binding cleft via loop 1reorientation). The Met side chain sulfur atom engages in a weak,favorable contact with the side chain guanidinium of Arg 230, while itsCε atom packs against the P238 pyrrolidine. Finally, the side chain ofthe inhibitor Leu sits slightly above the backbone of loop 2 (residues167-180), which is the steric boundary at this end of the substratebinding cleft. The hydrophobic Leu side chain is accommodated by weak,but favorable hydrophobic contacts with the side chain of V171, whilethe carbonyl oxygen of the amide bond between the inhibitor Met and Leuresidues engages in a weak hydrogen bond with the backbone amidenitrogen of V171. The terminal amide of the inhibitor faces the solvent.The docked model of mpp-RATKML was found to be highly desolvated.

4. Designing more Potent Small Molecule Inhibitors

Pharmacophore components were mapped to the docked conformation ofmpp-RATKML and it was found that the pseudo-peptide and small moleculeinhibitors share common structural/functional group features that conferbinding. Thus, incorporating features from the pseudo-peptide bindingmode may enhance the potencies of small molecule inhibitors.

For example, the mpp phenyl substituent, is a sterically andhydropathically superior match for binding subsite 1 compared to smallerhydrophobic moieties (i.e., chloro, methyl, and methoxy substituents).This indicates that aromatic heterocycles or planar, conjugatedguanidine/amidine/amide functional groups might also engage in favorableπ-π or cation-π interactions with Phe residues in this subsite.

The binding conformation of the inhibitor's Lys residue identifies apotential new hydrogen bonding contact, while the position of the dockedMet residue indicates new contacts and steric space that might beexploited. Finally, empirical results from deletion/additionexperiments, corroborated by data from the mpp-RATKML binding mode,suggest that there is an optimal length for BoNT/A LC inhibitors. If aninhibitor is too short (for example, mpp-RAT), it does not occupy enoughof the cleft, and consequently is not as potent; if it is too long (forexample, mpp-RATKMLGSG), components that fit in the cleft do notcontribute to activity. For the docked conformation of mpp-RATKML, thedistance between the mpp phenyl and the terminal Leu is about 23 Å.Thus, preferred compounds of the present invention are about 15 Å toabout 23 Å. Since amide bonds serve as mpp-RATKML pharmacophore planes,the compounds of the present invention may include bioisosteres such ascarbenes, imines, and azo linkages.

5. Database Searches of Candidate Compounds

An initial three-dimensional search query incorporating all of theoriginal pharmacophore components, in addition to the suggestedcomponents and criteria indicated above, identified no database hits.Consequently, combination search queries composed of 4 to 5pharmacophore components were conducted. Of the numerous queries thatwere generated, the one that identified the four potent inhibitorsincluded: (1) a pharmacophore component C that incorporated aromaticsubstituents (with and without heteroatoms), and planar, conjugatedpositive-ionizable functional groups; (2) a positive ionizablecomponent, labeled F, to mimic the Nζ nitrogen of the mpp-RATKML Lys;(3) an increased distance range between pharmacophore planes A and B(about 6.5 to about 13 Å); (4) the inclusion of carbenes, imines,amides, and azo linkages as pharmacophore planes; and (5) a totalcompound length constraint of about 23 Å. The structures and K_(i)values (determined in vitro) of the four inhibitors are shown in FIG. 1.

Molecular docking of the new inhibitors showed that they fit with goodsteric and hydropathic complementarity in the conformation of the BoNT/ALC that was used for mpp-RATKML docking For NSC 341909, 308574, and240898: (1) binding occurs down the length of the substrate cleft; (2)loop 1 serves as a solvent shield; (3) the planar, positive ionizable Ccomponents wedge between the side chain phenyls of Phe 162 and Phe193,with each engaging in a cation-7c interaction with the side-chain phenylof Phe 193; (4) the positive ionizable F components engage in hydrogenbonds with the side chain carboxylate of Glu 54 (this is one of thehydrogen bonding interactions predicted for the Lys Nζ of dockedmpp-RATKML, and with the side chain carboxylate of Glu 63, which, in theabsence of a substituent mimicking the mpp-RATKML Arg guanidinium(pharmacophore component E, is free to turn in the opposite direction;and (5) a biaryl associated heteroatom is positioned such that it mayinterfere with the zinc catalytic engine.

Activity of Candidate Compounds 1. NSC 240898 Inhibits SNAP-25 Cleavageby BoNT/A in Living Neurons

The candidate compounds were assayed for neuronal uptake, toxicity, andprotection. Chick spinal motor neurons were cultured by methods known inthe art. See Kuhn, T B (2003) Methods. Cell Biol. 71:67-87, which isherein incorporated by reference. In brief, fertilized chicken eggs(SPAFAS/Charles River Laboratories, North Franklin, Conn.) wereincubated at 37° C. for 6 days. Embryos were removed from the eggs andventral spinal cords were isolated from the embryos. Cells weredissociated by trypsinization and trituration. Cells were preplated intoa culture dish with Dullbecco's modified Eagle's medium plus 10% fetalbovine serum (FBS) for 1 hour to allow non-neuronal cells to attach tothe dish, thereby increasing the percentage of neuronal cells in thesuspension. Cells were centrifuged and resuspended in Leibovitz L15medium (Gibco/Invitrogen, Carlsbad, Calif.) with N3 supplement and 10%FBS. The mitotic inhibitor 5-fluorodeoxyuridine was added to furtherreduce the population of non-neuronal (i.e. dividing) cells. Cells wereplated into 6-well tissue culture plates that were coated first withpoly-L-lysine, then with laminin. Cultures were incubated overnight at37° C. prior to intoxication.

Autofluorescence was used to examine inhibitor entry into cells. Chickspinal motor neurons were incubated for 30 minutes with 20 μM inhibitorand examined for an increase in intracellular fluorescence on aninverted Nikon TE300 microscope with a standard DAPI filter set. Imageswere collected with a BioRad Radiance 2000 MP confocal/multiphotonsystem (Zeiss, Thornwood, N.Y.). For confocal images used to assaymorphological indicators of general cell health after toxin andinhibitor treatments, spinal motor neuron cultures were fixed for 30minutes in 3.7% formaldehyde and permeabilized in 0.2% Triton-X-100 for10 minutes. After blocking for 30 minutes in 1% bovine serum albumin(BSA), cells were incubated with 1:500 DM1A anti-α-tubulin (Sigma, St.Louis). Cells were then labeled with 1:500 Alexa 488 conjugated goatanti-mouse secondary antibody, 1:25 Texas red phalloidin, and Hoechststain (Molecular Probes, Eugene, Oreg.).

NSC 341909, 308574, and 240898 were found to become concentrated withinthe cells in about 30 minutes.

Next, potential cytotoxic effects of the candidate compounds wereassayed by incubating the cells with various concentrations of the NSC341909, 308574, and 240898 for about 3.5 hours. Specifically, cells werepre-incubated with a candidate compound for 45 minutes, followed by 3.5hours incubation with 10 nM BoNT/A (MetaBiologics Inc., Madison, Wis.)and the candidate compound. Cells were rinsed with fresh culture medium,scraped, collected, washed with PBS, lysed, and assessed for proteincontent using the Bradford protein assay (BioRad, Hercules, Calif.).Cell lysates were run on a 12% Tris-Glycine gel (Invitrogen, Carlsbad,Calif.), transferred to nitrocellulose, and probed with SMI 81 mouseanti-SNAP-25 (Sternberger Monoclonals Incorporated, Lutherville, Md.),and mouse anti-GAPDH (Covance Research Products, Inc., Berkeley, Calif.)primary antibodies. An HRP-conjugated goat anti-mouse secondary antibodywas used (Pierce, Rockford, Ill.) in combination with an ECL Westernblotting detection system (Pierce, Rockford, Ill.). Densitometry wasperformed using a UN-SCAN-IT gel automated digitizing system (SilkScientific, Inc, Orem, Utah).

While NSCs 341909 and 308574 proved to be highly cytotoxic atconcentrations as low as about 1 to about 5 μM, cells tolerated NSC240898 at concentrations as high as about 40 μM. Specifically, as shownin FIG. 2A, cells treated with 40 μM NSC 240898 for about 3.5 hours,fixed, and stained to show tubulin, actin filaments, and DNA revealedlittle or no morphological signs of damage, e.g. collapsed growth cones,abnormal axonal varicosities, or blebbing.

Pre-incubation of cells for about 30 to about 45 minutes with varyingconcentrations of NSC 240898, followed by intoxication with 10 nM BoNT/Ain the continued presence of inhibitor, demonstrated a robustdose-dependent inhibition of SNAP-25 cleavage in Western blot analysesas shown in FIG. 2B and FIG. 2C. Thus, compounds of the presentinvention, such as NSC 240898 may be used to treat, prevent or inhibitBoNT/A intoxication in a subject.

Isothermal Titration Calorimetry

To confirm the specific nature of the NSC 240898:BoNT/A LC interaction,isothermal titration calorimetry (ITC) studies were performed usingmethods known in the art. Specifically, purified BoNT/A LC was subjectedto a final gel filtration step (Superdex 200 column) to ensure acomplete buffer exchange, as well as to exclude trace amounts ofautocleavage products. All experiments were carried out in the samebuffer to control for heat of dilution effects: 50 mM Hepes (pH 7.4)supplemented with 150 mM NaCl and 0.5 mM ZnCl₂. Concentrations of NSC240898 solutions were confirmed by UV/Vis absorbance measurements.Calorimetric titration was performed multiple times on a VP-ITCcalorimeter (MicroCal, Northhampton, Mass.) at 293 K. BoNT/A LC was usedat a concentration of 28 μM in the ITC cell and inhibitor NSC 240898 ata concentration of 390 μM in the injection syringe. Prior to thetitration, the samples were degassed for 10 minutes. The positivedeflections observed at the end of the titration reflect the enthalpy ofdilution of the inhibitor solution and were subtracted from the bindingdata. The first injection systematically showed a decreased enthalpy dueto the technical limitations of the instrument and was omitted fromcurve fitting. The titration curve was then fit to a single site modelby non-linear least squares regression as implemented by MicroCalSoftware Inc. in the Origin software package. According to this model,the parameters to be determined are: the number of binding sites (N),the binding enthalpy per site (AH), and the binding constant (K_(d)).

Table 2 shows measured and calculated thermodynamic values of NSC 240898binding to the BoNT/A LC. The stoichiometry parameter (N) was adjustedin the fit in order to account for uncertainties in the concentration ofprotein in solution.

TABLE 2 Temperature (° C.) 20 N 0.83 ± 0.04 K_(a) (M⁻¹) 2.18 × 10⁵ ± 4.5× 10⁴ K_(d) (μM) 4.6 ΔH (kcal mol⁻¹) −3.04 ± 0.20 ΔS (cal mol⁻¹ K⁻¹)14.0

Representative data are shown in FIGS. 3A-3B. A low affinity bindingevent (K_(d)˜4.6 μM) that was largely entropy-driven (ΔS=14 cal/mol K)was observed; comparatively, the enthalpic contribution was relativelylow (ΔH=−3.04 kcal/mol). Thus, affinity of the small molecule compoundsof the present invention may be significantly improved by optimizingelectrostatic interactions with surrounding enzyme residues.

Due to the low affinity of the interaction, a large excess of NSC 240898was necessary to drive the titration to saturation. While enzymaticassays and molecular dynamics simulations indicate the number of NSC240898 binding sites to be one, this parameter was adjusted in the fitin order to account for uncertainties in the concentration of protein insolution. Subsequently, N=0.83±0.04 was obtained, which is consistentwith a 1:1 stoichiometry (along with a small fraction of misfoldedprotein as often observed in ITC experiments).

The significant entropic contribution observed suggests a burial ofhydrophobic surfaces and the release of solvent upon inhibitor binding,consistent with the hydrophobic nature of NSC 240898. Taking intoaccount the conformational variability of loops 1, 3 and 4 of the BoNT/ALC this solvent release is probably accompanied by a conformationalchange in at least one of the flexible loops.

NSC 328398

NSC 328398 is a novel scaffold that was discovered using a differentthree-dimensional search query that was based on the refinedpharmacophore, and exhibited 45% inhibition of BoNT/A LC metalloproteaseactivity at 10 μM.

New Small Molecule Compounds

Other small molecule compounds which were identified according to themethods disclosed herein to inhibit BoNT/A LC metalloprotease activityare provided in Table 3.

TABLE 3 NSC Structure % Inhibition 341909

68% 308574

63% 240808

61% 341907

60% 266472

57% 330690

53% 278999

53% 308571

52% 290107

52% 290108

51% 294200

48% 317884

47% 294203

46% 294494

45% 317881

45% 330688

43% 317886

43% 317883

41% 352341

40% 294204

38% 341911

37% 300511

37% 607617

36% 294202

35% 317880

35%

Thus, compounds of the present invention have the following tructuralformula:

wherein

n is 1 or 2;

X¹, X², X³, X⁴, X⁵ and X⁶ are each independently N, S, O, SO₂, CR⁷ orNR⁸ and at least one of X¹ or X² is N, S, O, SO₂, or NR⁸;

L is a linker which may be a direct bond or

where Z is an optionally substituted alkyl, alkenyl, dialkenyl,trialkenyl, or aryl, or C(O)NH; and

R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are each independently hydrogen, amino,amine with stabilized carbocations, carboxyl, optionally substitutedalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,alkoxy, aryoxy, cycloalkoxy, heteroaryloxy, alkoxycarbonyl, alkylamino,carbamoyl, alkylaminocarbonyl, alkylsulfhydryl, alkylhydroxymate; and

R⁸ is hydrogen, OH, a halogen, or an optionally substituted alkyl.

It is noted that in the structural formulas of the present invention,the bond orders of the specified rings may vary when the variousheteroatoms introduce specific requirements to satisfy aromaticity,prevent antiaromaticity, and stabilize tautomeric forms due tolocalization. Thus, the appropriate bond orders of the ring structuresin the structural formulas of the present invention are contemplatedherein.

In some embodiments, R¹ is hydrogen, amidine, 2-imidazoline, amino,guanidine, methyl, aminomethyl-hydroxamine, or methylamine-guanidine.

In some embodiments, R² is hydrogen, amidine, 2-imidazoline, amino,guanidine, methyl, aminomethyl-hydroxamine, or methylamine-guanidine.

In some embodiments, R³ is hydrogen, amidine, 2-imidazoline, amino,guanidine, methyl, aminomethyl-hydroxamine, or methylamine-guanidine.

In some embodiments, R⁴ is hydrogen, amidine, 2-imidazoline, amino,guanidine, methyl, aminomethyl-hydroxamine, or methylamine-guanidine.

In some embodiments, R⁵ is hydrogen, amidine, 2-imidazoline, amino,guanidine, methyl, aminomethyl-hydroxamine, methylamine-guanidine,4-oxy-benzamidine, 1H-indole-6-caboxamidine, or1H-indole-5-carboxamidine.

In some embodiments, R⁶ is hydrogen, amidine, benzamidine,benzimidazoline, imidazoline, guanidine, imidazole, oxazole,benzofuran-2-yl-imidazoline, benzofuran-2-yl-amidine,benzofuran-2-yl-guanidine, benzothiophene-2-yl-imidazoline,benzothiophene-2-yl-amidine, benzene-2-yl-amidine,benzofuran-2-yl-imidazole, or benzofuran-2-yl-oxazole.

In some embodiments, X¹ is N, NH, S, O, SO₂, CH, C—CH₃, C-phenyl,N-ethanol, N-chloroethyl, C-amino, C-(2-indole-6-imidazoline),C-(2-indole-6-amidine), C-(2-indole-5-imidazoline), orC-(2-indole-5-amidine).

In some embodiments, X² is N, NH, S, O, SO₂, CH, C—CH₃, C-phenyl,N-ethanol, N-chloroethyl, C-amino, C-(2-indole-6-imidazoline),C-(2-indole-6-amidine), C-(2-indole-5-imidazoline), orC-(2-indole-5-amidine).

In some embodiments, X³ is N, NH, S, O, SO₂, or CH.

In some embodiments, X⁴ is N, NH, S, O, SO₂, or CH.

In some embodiments, X⁵ is N, NH, S, O, SO₂, or CH.

In some embodiments, X⁶ is N, NH, S, O, SO₂, or CH.

In some embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶ or R⁷ is —H,—CH₃,

In some embodiments, R⁵ is —NH₂,

In some embodiments, R⁶ is

In some embodiments, R⁷ is —H, —CH₃, —NH₂,

In some embodiments, R⁸ is —H, —(CH₂)₂OH, or —(CH₂)₂Cl.

In some embodiments, L is a direct bond,

In some embodiments, compounds of the present invention have thefollowing structural formulae:

wherein

n is 1 or 2;

X¹, X², X³, X⁴, X⁵ and X⁶ are each independently N, S, O, SO₂, CR⁷ orNR⁸ and at least one of X¹ or X² is N, S, O, SO₂, or NR⁸;

L is a linker which may be a direct bond or

where Z is an optionally substituted alkyl, alkenyl, dialkenyl,trialkenyl, or aryl, or C(O)NH; and

R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are each independently hydrogen, amino,amine with stabilized carbocations, carboxyl, optionally substitutedalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,alkoxy, aryoxy, cycloalkoxy, heteroaryloxy, alkoxycarbonyl, alkylamino,carbamoyl, alkylaminocarbonyl, alkylsulfhydryl, alkylhydroxymate; and

R⁸ is hydrogen, OH, a halogen, or an optionally substituted alkyl.

In some embodiments, the compound is NSC 92833, NSC 103699, NSC 103701,NSC 130681, NSC 240890, NSC 240891, NSC 240893, NSC 240894, NSC 240895,NSC 240896, NSC 240897, NSC 240898, NSC 240899, NSC 240900, NSC 266472,NSC 266474, NSC 266475, NSC 266476, NSC 266477, NSC 266482, NSC 278995,NSC 278996, NSC 278997, NSC 278999, NSC 290107, NSC 290108, NSC 290109,NSC 290111, NSC 291103, NSC 294199, NSC 294200, NSC 294201, NSC 294202,NSC 294203, NSC 294204, NSC 294206, NSC 294207, NSC 294208, NSC 294494,NSC 300509, NSC 300510, NSC 300511, NSC 300512, NSC 302569, NSC 308569,NSC 308570, NSC 308571, NSC 308572, NSC 308573, NSC 308574, NSC 317880,NSC 317881, NSC 317883, NSC 317884, NSC 317885, NSC 317886, NSC 317887,NSC 328398, NSC 330687, NSC 330688, NSC 330689, NSC 330690, NSC 341082,NSC 341907, NSC 341909, NSC 341910, NSC 341911, NSC 352341, NSC 369718,NSC 369721, NSC 607617, or NSC 12155. The structural formulas of thesecompounds are known in the art and may be obtained from various sourcesincluding the World Wide Web atdtp.nci.nih.gov/dtpstandard/ChemData/index.jsp andncbi.nlm.nih.gov/entrez/query.fcgi?CMD=&DB=PubMed and are hereinincorporated by reference.

Derivatives of NSC 240898 and Synthesis Thereof

Compounds of the present invention may be synthesized using methodsknown in the art. For example, compounds which are structurally similarto NSC 240898, such as those shown in Table 1, may be synthesized asprovided herein.

TABLE 1

No. R^(a) and R^(b) X Y MW CLogP HBA HBD DRF 14 CN NH CH 335.36 5.58 4 15 15 CN NH N 336.35 4.35 5 1 5 16 CN O CH 352.41 6.31 3 0 5 17 CN O N353.40 5.04 4 0 5 18 CONH₂ NH CH 371.39 3.55 4 3 5 19 CONH₂ NH N 372.382.31 5 3 5 20 CONH₂ O CH 388.44 4.52 3 2 5 21 CONH₂ O N 389.43 3.25 4 25 22 C(═NH)NH₂ NH CH 369.42 3.39 6 5 5 23 C(═NH)NH₂ NH N 370.41 2.21 7 55 24 C(═NH)NH₂ O CH 386.47 4.47 5 4 5 25 C(═NH)NH₂ O N 387.46 3.21 6 4 5

Also recorded in Table 1 are Lipinski's “drug-like” guidelines for thesecompounds; molecular weight <500 kda, low lipophilicity (cLogP<5), lessthan 5 hydrogen bond donors, less than 10 hydrogen bond acceptors. SeeLipinski, et al. (2001) Adv. Drug. Deliv. Rev. 46:3-26, which is hereinincorporated by reference.

The compounds in Table 1 may be synthesized according to Scheme A, partsA-D as follows:

Scheme 1. Specific Example of the Synthesis of an NCS 240898 Analog A.Pd-Catalyzed Aromatic Substitution

B. Indole Synthesis with Intermediates 3 and 6

C. Benzothiophene Synthesis

D. R Group Transformations

As shown in Scheme 1, Part A, methyl 4-bromobenzoate (1) is treated with4-cyanophenol in the presence of a palladium (II) catalyst to producethe diphenyl ether 2, which is then hydrolyzed to give4-(4-cyanophenoxy)benzoic acid (3). See Aranyos et al. (1999) J Am ChemSoc 121:4369; and Mann et al. (1999) J Am Chem Soc 121:3224, which areherein incorporated by reference. Compound 3 can then serve as a partnerwith 3-amino-4-methylbenzoic acid (7) to provide the necessary substratefor a modified Madelung indole synthesis (Part B). Esterification of 7followed by coupling with benzoic acid 3 will provide benzamide 8a,which, upon treatment with dimethylaluminum amide, will give thecorresponding nitrile 9a. See Theodre & Nelson (1987) J Org Chem52:1309; and Wood et al. (1979) Tetrahedron Lett 20:4907-4910, which areherein incorporated by reference. Exposure of 9a to lithiumdiisopropylamide will then effect a dehydrative cyclization to producethe NSC 240898 analog, e.g. compound 10a. See Houlihan et al. (1981) JOrg Chem 46:4511, 4515, which is herein incorporated by reference.Likewise, analog 10b can be synthesized in an analogous fashion, asindicated in Parts A and B. Note that the addition of a nitrogen atom inthe central ring may enhance zinc binding, since the site ofcoordination for the zinc will now become bidentate. An alternative planto produce compounds 10, not shown here, is to use a Fischer indolesynthesis protocol. See Robinson, B. (1983) The Fischer IndoleSynthesis. Wiley, New York; and Sundberg, R. J. (1970) The Chemistry ofIndoles. Academic Press, New York, pp. 142-163, which are hereinincorporated by reference.

Synthesis of one example of the proposed inhibitors shown in theSynthetic Analysis, where X═S and Y═N is also shown in Scheme 1 (PartC). 6-Cyanobenzo[b]thiophene (11) is coupled with 6-chloro-3-pyridinolto produce intermediate 12, using either an iridium catalyst andbis(pinacolato)boron to prepare 6-cyanobenzo[b]thiophene-2-boronic acid,and then using this boronic acid in a palladium catalyzed Suzukicoupling with 6-chloro-3-pyridinol; or, by generating the anion at the2-position of 11 with lithium diisopropylamide and quenching this anionwith 6-chloropyridin-3-ol. See U.S. Patent Publication 20050148775; andGuiles et al. (1996) J Org Chem 61:5169, which are herein incorporatedby reference. Subsequent conversion of intermediate 12 to targetcompound 13 can then be accomplished by nucleophilic aromaticsubstitution, by treating the anion of 12 with 4-fluorobenzonitrile.Note that compound 13, where Y═CH, can be prepared in an analogousfashion. Since sulfur is a very good coordinating ligand for zinc, it isexpected that compound 13 and compounds derived from compound 13 couldprovide excellent inhibitors of the BoNT/A LC metalloproteinase.

Compounds of general structure 10 and 13 are very versatile compounds.In addition to being target compounds themselves, they are portals tothe preparation of a wide variety of additional compounds. As shown inScheme 1, Part D, the dinitriles can readily be converted to thediacids, by hydrolysis under acidic conditions. These diacids are alsovery versatile, dual point compounds. The diacids will be converted tothe simple diamides by preparing the diacid chlorides and quenching themwith ammonia. Alternatively, if one chooses to avoid the diacids asdiscreet intermediates, we will treat the dinitriles directly withsulfuric acid or with potassium hydroxide to hydrate the nitrile groupsand produce the diamides in one step. See Sarel & Newman (1956) J AmChem Soc 78:5416; and Hall & Gisler (1976) J Org Chem 41:3769, which areherein incorporated by reference. The diamides, in turn, will beconverted to the diamidines by treatment with Lawessen's reagentfollowed by S-methylation, and subsequent treatment of these activatedintermediates with ammonia. See Yde, et al. (1984) Tetrahedron Lett40:2047, which is herein incorporated by reference. Note that all of thegeneral structures shown in Scheme 1 are quite versatile in theirreactivities and potential transformations, and the transformationsshown are only a selected few from those transformations that can beperformed. Also note that specific compounds chosen for display in Table1 are all covered by the general structures.

Preferred compounds of the present invention also include those whichare structurally related to NSC341909, NSC 308574, and Q2-15, such asthe following:

In accordance with a convention used in the art,

is used in structural formulas herein to depict the bond that is thepoint of attachment of the moiety or substituent to the core or backbonestructure.

Where chiral carbons are included in chemical structures, unless aparticular orientation is depicted, both sterioisomeric forms areintended to be encompassed.

A “halo” or “halogen” means fluorine, bromine, chlorine, and iodine.

An “alkyl” is intended to mean a straight or branched chain monovalentradical of saturated and/or unsaturated carbon atoms and hydrogen atoms,such as methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl(n-Bu), isobutyl (i-Bu), t-butyl (t-Bu), (sec-Bu), and the like, whichmay be unsubstituted (i.e., contain only carbon and hydrogen) orsubstituted by one or more suitable substituents as defined below. A“lower alkyl group” is intended to mean an alkyl group having from 1 to8 carbon atoms in its chain.

A “haloalkyl” refers to an alkyl that is substituted with one or moresame or different halo atoms, e.g., —CH₂Cl, —CF₃, —CH₂CF₃, —CH₂CCl₃, andthe like.

An “alkenyl” means straight and branched hydrocarbon radicals havingfrom 2 to 8 carbon atoms and at least one double bond such as ethenyl,3-buten-1-yl, 2-ethenylbutyl, 3-hexen-1-yl, and the like. The term“alkenyl” includes, cycloalkenyl, and heteroalkenyl in which 1 to 3heteroatoms selected from O, S, N or substituted nitrogen may replacecarbon atoms.

An “alkynyl” means straight and branched hydrocarbon radicals havingfrom 2 to 8 carbon atoms and at least one triple bond and includes, butis not limited to, ethynyl, 3-butyn-1-yl, propynyl, 2-butyn-1-yl,3-pentyn-1-yl, and the like.

A “cycloalkyl” is intended to mean a non-aromatic monovalent monocyclicor polycyclic radical having from 3 to 14 carbon atoms, each of whichmay be saturated or unsaturated, and may be unsubstituted or substitutedby one or more suitable substituents as defined herein, and to which maybe fused one or more aryl groups, heteroaryl groups, cycloalkyl groups,or heterocycloalkyl groups which themselves may be unsubstituted orsubstituted by one or more substituents. Examples of cycloalkyl groupsinclude cyclopropyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclobutyl,adamantyl, norpinanyl, decalinyl, norbornyl, cyclohexyl, andcyclopentyl.

A “heterocycloalkyl” is intended to mean a non-aromatic monovalentmonocyclic or polycyclic radical having 1-5 heteroatoms selected fromnitrogen, oxygen, and sulfur, and may be unsubstituted or substituted byone or more suitable substituents as defined herein, and to which may befused one or more aryl groups, heteroaryl groups, cycloalkyl groups, orheterocycloalkyl groups which themselves may be unsubstituted orsubstituted by one or more substituents. Examples of heterocycloalkylgroups include oxiranyl, pyrrolidinyl, piperidyl, tetrahydropyran, andmorpholinyl.

An “aryl” (Ar) is intended to mean an aromatic monovalent monocyclic orpolycyclic radical comprising generally between 5 and 18 carbon ringmembers, which may be unsubstituted or substituted by one or moresuitable substituents as defined herein, and to which may be fused oneor more cycloalkyl groups, heterocycloalkyl groups, or heteroarylgroups, which themselves may be unsubstituted or substituted by one ormore suitable substituents. Thus, the term “aryl group” includes abenzyl group (Bzl). Examples include phenyl, biphenyl,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, and phenanthryl.

A “heteroaryl” is intended to mean an aromatic monovalent monocyclic orpolycyclic radical comprising generally between 4 and 18 ring members,including 1-5 heteroatoms selected from nitrogen, oxygen, and sulfur,which may be unsubstituted or substituted by one or more suitablesubstituents as defined below, and to which may be fused one or morecycloalkyl groups, heterocycloalkyl groups, or aryl groups, whichthemselves may be unsubstituted or substituted by one or more suitablesubstituents. Examples include thienyl, furanyl, thiazolyl, triazolyl,imidazolyl, isoxazolyl, oxadiazolyl, tetrazolyl, pyridyl, pyrrolyl,thiadiazolyl, oxadiazolyl, oxathiadiazolyl, thiatriazolyl, pyrimidinyl,isoquinolinyl, quinolinyl, napthyridinyl, phthalimidyl, benzimidazolyl,and benzoxazolyl.

A “hydroxy” is intended to mean the radical —OH.

An “alkoxy” is intended to mean the radical —OR, where R is an alkylgroup. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, and thelike.

A “hydroxyalkyl” means an alkyl that is substituted with one, two, orthree hydroxy groups, e.g. hydroxymethyl, 1 or 2-hydroxyethyl, 1,2-,1,3-, or 2,3-dihydroxypropyl, and the like.

A “haloalkoxy” refers to an —O-(haloalkyl) group. Examples includetrifluoromethoxy, tribromomethoxy, and the like.

A “cycloalkoxy” is intended to mean the radical —OR, where R isacycloalkyl or heterocycloalkyl group.

An “aryloxy” is intended to mean the radical —OR, where R is an aryl orheteroaryl group. Examples include phenoxy, pyridinyloxy, furanyloxy,thienyloxy, pyrimidinyloxy, pyrazinyloxy, and the like.

An “acyl” is intended to mean a —C(O)—R radical, where R is an alkyl oraryl, bonded through a carbonyl group. Acyl groups include acetyl,benzoyl, and the like.

An “aralkyl” means an alkyl that is substituted with an aryl group.Examples include —CH₂-phenyl, —(CH₂)₂-phenyl, —(CH₂)₃-phenyl,—CH₃CH(CH₃)CH₂-phenyl, and the like.

A “heteroaralkyl” group means an alkyl that is substituted with aheteroaryl group. Examples include —CH₂-pyridinyl, —(CH₂)₂-pyrimidinyl,—(CH₂)₃-imidazolyl, and the like.

A “carboxy” is intended to mean the radical —C(O)OH.

An “alkoxycarbonyl” is intended to mean the radical —C(O)OR, where R isan alkyl group. Examples include methoxycarbonyl, ethoxycarbonyl, andthe like.

An “amino” is intended to mean the radical —NH₂.

An “amine with stabilized carbocations” are comprised of two or more NH₂groups that contribute lone pairs to configure a highly stabilizedcarbocation. Examples include amidines and guanidines.

An “alkylamino” is intended to mean the radical —NHR, where R is analkyl group or the radical —NR^(a)R^(b), where R^(a) and R^(b) are eachindependently an alkyl group. Examples of alkylamino groups includemethylamino, ethylamino, n-propylamino, isopropylamino, tert-butylamino,n-pentylamino, n-hexylamino, N,N-dimethylamino, N,N-diethylamino,N-ethyl-N-methylamino, N-methyl-N-n-propylamino,N-isopropyl-N-n-propylamino, N-t-butyl-N-methylamino,N-ethyl-N-n-pentylamino, N-n-hexyl-N-methylamino and the like.

An “alkylsulfhydryl” is intended to mean R—SH, where R is an alkylgroup.

Examples include methylsulfhydryl, ethylsulfhydryl, n-propylsulfhydryl,iso-propylsulfhydryl, n-butylsulfhydryl, iso-butylsulfhydryl,secondary-butylsulfhydryl, tertiary-butylsulfhydryl. Preferablealkylsulfhydryl groups are methylsulfhydryl, ethylsulfhydryl,n-propylsulfhydryl, n-butylsulfhydryl, and the like.

An “alkylhydroxymate” is intended to mean the radical R—C(O)NH—OH, whereR is an alkyl group. Examples include methylhydroxymate,ethylhydroxymate, n-propylhydroxymate, iso-propylhydroxymate,n-butylhydroxymate, iso-butylhydroxymate, secondary-butylhydroxymate,tertiary-butylhydroxymate. Preferable alkylhydroxymate groups aremethylhydroxymate, ethylhydroxymate, n-propylhydroxymate,n-butylhydroxymate, and the like. A “carbamoyl” is intended to mean theradical —C(O)NH₂.

A “carbamoyl” is intended to mean the radical —C(O)NH₂.

An “alkylaminocarbonyl” is intended to mean the radical —C(O)NHR, whereR is an alkyl group or the radical —C(O)NR^(a)R^(b), where R^(a) andR^(b) are each independently an alkyl group. Examples includemethylaminocarbonyl, ethylaminocarbonyl, dimethylaminocarbonyl,methylethylaminocarbonyl, and the like.

A “mercapto” is intended to mean the radical —SH.

An “alkylthio” is intended to mean the radical —SR, where R is an alkylor cycloalkyl group. Examples of alkylthio groups include methylthio,ethylthio, n-propylthio, isopropylthio, tert-butylthio, n-pentylthio,n-hexylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio,cyclohexylthio, and the like.

An “arylthio” is intended to mean the radical —SR, where R is an aryl orheteroaryl group. Examples include phenylthio, pyridinylthio,furanylthio, thienylthio, pyrimidinylthio, and the like.

A “thioacyl” is intended to mean a —C(S)—R radical, where R is an alkylor aryl, bonded through a thiol group.

An “alkylsulfonyl” is intended to mean the radical —SO₂R, where R is analkyl group. Examples include methylsulfonyl, ethylsulfonyl,n-propylsulfonyl, iso-propylsulfonyl, n-butylsulfonyl,iso-butylsulfonyl, secondary-butylsulfonyl, tertiary-butylsulfonyl.Preferable alkylsulfonyl groups are methylsulfonyl, ethylsulfonyl,n-propylsulfonyl, n-butylsulfonyl, and the like.

A “leaving group” (Lv) is intended to mean any suitable group that willbe displaced by a substitution reaction. One of ordinary skill in theart will know that any conjugate base of a strong acid can act as aleaving group. Illustrative examples of suitable leaving groups include,but are not limited to, —F, —Cl, —Br, alkyl chlorides, alkyl bromides,alkyl iodides, alkyl sulfonates, alkyl benzenesulfonates, alkylp-toluenesulfonates, alkyl methanesulfonates, triflate, and any groupshaving a bisulfate, methyl sulfate, or sulfonate ion.

A “protecting group” is intended to refer to groups that protect one ormore inherent functional group from premature reaction. Suitableprotecting groups may be routinely selected by those skilled in the artin light of the functionality and particular chemistry used to constructthe compound. Examples of suitable protecting groups are described, forexample, in Greene and Wuts, Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley and Sons, New York, N.Y. (1999).

The term “suitable organic moiety” is intended to mean any organicmoiety recognizable, such as by routine testing, to those skilled in theart as not adversely affecting the inhibitory activity of the inventivecompounds. Illustrative examples of suitable organic moieties include,but are not limited to, hydroxyl groups, alkyl groups, oxo groups,cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroarylgroups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups,alkoxyl groups, carboxyl groups, amino groups, alkylamino groups,dialkylamino groups, carbamoyl groups, arylthio groups, heteroarylthiogroups, and the like.

In general, the various moieties or functional groups for variables inthe formulae may be “optionally substituted” by one or more suitable“substituents”. The term “substituent” or “suitable substituent” isintended to mean any suitable substituent that may be recognized orselected, such as through routine testing, by those skilled in the art.Illustrative examples of useful substituents are those found in theexemplary compounds that follow, as well as a halogen; C₁₋₆-alkyl;C₁₋₆-alkenyl; C₁₋₆-alkynyl; hydroxyl; C₁₋₆ alkoxyl; amino; nitro; thiol;thioether; imine; cyano; amido; phosphonato; phosphine; carboxyl;carbonyl; aminocarbonyl; thiocarbonyl; sulfonyl; sulfonamine;sulfonamide; ketone; aldehyde; ester; oxygen (═O); haloalkyl (e.g.,trifluoromethyl); carbocyclic cycloalkyl, which may be monocyclic orfused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl), or a heterocycloalkyl, which may bemonocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, or thiazinyl); carbocyclic orheterocyclic, monocyclic or fused or non-fused polycyclic aryl (e.g.,phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl,pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl,pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); amino(primary, secondary, or tertiary); nitro; thiol; thioether, O-loweralkyl; O-aryl, aryl; aryl-lower alkyl; CO₂CH₃; CONH₂; OCH₂CONH₂; NH₂;SO₂NH₂; OCHF₂; CF₃; OCF₃; and the like. Such moieties may also beoptionally substituted by a fused-ring structure or bridge, for exampleOCH₂—O. All of these substituents may optionally be further substitutedwith a substituent selected from groups such as hydroxyl groups,halogens, oxo groups, alkyl groups, acyl groups, sulfonyl groups,mercapto groups, alkylthio groups, alkyloxyl groups, cycloalkyl groups,heterocycloalkyl groups, aryl groups, heteroaryl groups, carboxylgroups, amino groups, alkylamino groups, dialkylamino groups, carbamoylgroups, aryloxyl groups, heteroaryloxyl groups, arylthio groups,heteroarylthio groups, and the like.

The term “optionally substituted” is intended to expressly indicate thatthe specified group is unsubstituted or substituted by one or moresuitable substituents, unless the optional substituents are expresslyspecified, in which case the term indicates that the group isunsubstituted or substituted with the specified substituents. As definedabove, various groups may be unsubstituted or substituted (i.e., theyare optionally substituted) unless indicated otherwise herein (e.g., byindicating that the specified group is unsubstituted).

It is understood that while a compound of the general structuralformulas herein may exhibit the phenomenon of tautomerism, thestructural formulas within this specification expressly depict only oneof the possible tautomeric forms. It is therefore to be understood thatthe structural formulas herein are intended to represent any tautomericform of the depicted compound and is not to be limited merely to aspecific compound form depicted by the structural formulas.

It is also understood that the structural formulas are intended torepresent any configurational form of the depicted compound and is notto be limited merely to a specific compound form depicted by thestructural formulas.

Some of the compounds of the present invention may exist as singlestereoisomers (i.e., essentially free of other stereoisomers),racemates, or mixtures of enantiomers, diastereomers, or both when theycontain one or more stereogenic centers as designated by R or Saccording to the Cahn-Ingold-Prelog rules whether the absolute orrelative configuration is known. All such single stereoisomers,racemates and mixtures thereof are intended to be within the scope ofthe present invention.

Some of the compounds in the present invention may exist as geometricisomers as the result of containing a stereogenic double bond. In suchcases, they may exist either as pure or mixtures of cis or transgeometric isomers or (E) and (Z) designated forms according to theCahn-Ingold-Prelog rules and include compounds that adopt a double bondconfiguration as a result of electronic delocalization.

As generally understood by those skilled in the art, an optically purecompound having one or more chiral centers (i.e., one asymmetric atomproducing unique tetrahedral configuration) is one that consistsessentially of one of the two possible enantiomers (i.e., isenantiomerically pure), and an optically pure compound having more thanone chiral center is one that is both diastereomerically pure andenantiomerically pure. If the compounds of the present invention aremade synthetically, they may be used in a form that is at least 90%optically pure, that is, a form that comprises at least 90% of a singleisomer (80% enantiomeric excess (e.e.) or diastereomeric excess (d.e.),more preferably at least 95% (90% e.e. or d.e.), even more preferably atleast 97.5% (95% e.e. or d.e.), and most preferably at least 99% (98%e.e. or d.e.).

Additionally, the structural formulas herein are intended to cover,where applicable, solvated as well as unsolvated forms of the compounds.A “solvate” is intended to mean a pharmaceutically acceptable solvateform of a specified compound that retains the biological effectivenessof such compound. Examples of solvates include compounds of theinvention in combination with water, isopropanol, ethanol, methanol,dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, oracetone. Also included are miscible formulations of solvate mixturessuch as a compound of the invention in combination with an acetone andethanol mixture. In a preferred embodiment, the solvate includes acompound of the invention in combination with about 20% ethanol andabout 80% acetone. Thus, the structural formulas include compoundshaving the indicated structure, including the hydrated as well as thenon-hydrated forms.

As indicated above, the compounds of the invention also include activetautomeric and stereoisomeric forms of the compounds of the presentinvention, which may be readily obtained using techniques known in theart. For example, optically active (R) and (S) isomers may be preparedvia a stereospecific synthesis, e.g., using chiral synthons and chiralreagents, or racemic mixtures may be resolved using conventionaltechniques.

Additionally, the compounds of the invention include pharmaceuticallyacceptable salts, multimeric forms, prodrugs, active metabolites,precursors and salts of such metabolites of the compounds of the presentinvention.

The term “pharmaceutically acceptable salts” refers to salt forms thatare pharmacologically acceptable and substantially non-toxic to thesubject being treated with the compound of the invention.Pharmaceutically acceptable salts include conventional acid-additionsalts or base-addition salts formed from suitable non-toxic organic orinorganic acids or inorganic bases. Exemplary acid-addition saltsinclude those derived from inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid,phosphoric acid, and nitric acid, and those derived from organic acidssuch as p-toluenesulfonic acid, methanesulfonic acid, ethane-disulfonicacid, isethionic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic acid,acetic acid, phenylacetic acid, propionic acid, glycolic acid, stearicacid, lactic acid, malic acid, tartaric acid, ascorbic acid, maleicacid, hydroxymaleic acid, glutamic acid, salicylic acid, sulfanilicacid, and fumaric acid. Exemplary base-addition salts include thosederived from ammonium hydroxides (e.g., a quaternary ammonium hydroxidesuch as tetramethylammonium hydroxide), those derived from inorganicbases such as alkali or alkaline earth-metal (e.g., sodium, potassium,lithium, calcium, or magnesium) hydroxides, and those derived fromnon-toxic organic bases such as basic amino acids.

The term “multimer” refers to multivalent or multimeric forms of activeforms of the compounds of the invention. Such “multimers” may be made bylinking or placing multiple copies of an active compound in closeproximity to each other, e.g., using a scaffolding provided by a carriermoiety. Multimers of various dimensions (i.e., bearing varying numbersof copies of an active compound) may be tested to arrive at a multimerof optimum size with respect to binding site interactions. Provision ofsuch multivalent forms of active binding compounds with optimal spacingbetween the binding site moieties may enhance binding site interactions.See e.g. Lee et al., (1984) Biochem. 23:4255. The artisan may controlthe multivalency and spacing by selection of a suitable carrier moietyor linker units. Useful moieties include molecular supports comprising amultiplicity of functional groups that can be reacted with functionalgroups associated with the active compounds of the invention. A varietyof carrier moieties may be used to build highly active multimers,including proteins such as BSA (bovine serum albumin), peptides such aspentapeptides, decapeptides, pentadecapeptides, and the like, as well asnon-biological compounds selected for their beneficial effects onabsorbability, transport, and persistence within the target organism.Functional groups on the carrier moiety, such as amino, sulfhydryl,hydroxyl, and alkylamino groups, may be selected to obtain stablelinkages to the compounds of the invention, optimal spacing between theimmobilized compounds, and optimal biological properties.

“A pharmaceutically acceptable prodrug” is a compound that may beconverted under physiological conditions or by solvolysis to thespecified compound or to a pharmaceutically acceptable salt of suchcompound, or a compound that is biologically active with respect to theintended pharmacodynamic effect. “A pharmaceutically active metabolite”is intended to mean a pharmacologically active product produced throughmetabolism in the body of a specified compound or salt thereof. Prodrugsand active metabolites of a compound may be identified using routinetechniques known in the art. See, e.g., Bertolini, G. et al., (1997) J.Med. Chem. 40:2011-2016; Shan, D. et al., J. Pharm. Sci., 86(7):765-767;Bagshawe K., (1995) Drug Dev. Res. 34:220-230; Bodor, N., (1984)Advances in Drug Res. 13:224-331; Bundgaard, H., Design of Prodrugs(Elsevier Press, 1985); and Larsen, I. K., Design and Application ofProdrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds.,Harwood Academic Publishers, 1991).

If the compound of the present invention is a base, the desiredpharmaceutically acceptable salt may be prepared by any suitable methodavailable in the art, for example, treatment of the free base with aninorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyrvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an α-hydroxy acid, such as citric acid or tartaricacid, an amino acid, such as aspartic acid or glutamic acid, an aromaticacid, such as benzoic acid or cinnamic acid, a sulfonic acid, such asp-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the present invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include organic salts derived from basicamino acids, such as lysine and arginine, ammonia, primary, secondary,and tertiary amines, and cyclic amines, such as piperidine, morpholineand piperazine, and inorganic salts derived from sodium, calcium,potassium, magnesium, manganese, iron, copper, zinc, aluminum andlithium.

In the case of compounds that are solids, it is understood by thoseskilled in the art that the compound of the present invention and saltsmay exist in different crystal or polymorphic forms, all of which areintended to be within the scope of the present invention and specifiedstructural formulas.

The compounds of the present invention are useful in inhibiting BoNT/ALC metalloprotease activity. The compounds of the present invention arealso useful in treating, inhibiting or preventing intoxication caused bybotulinum toxin in a subject. Further, since some of the compounds ofthe present invention are found to exhibit antibacterial activity, thecompounds of the present invention are useful in inhibiting, reducing orpreventing growth of or destroying bacteria of at least one bacterialstrain.

The compounds of the present invention are also useful in treating,inhibiting or preventing an infection caused by bacterial of at leastone bacterial strain in a subject. The bacteria belong to various grampositive and gram negative bacteria strains including Bacillus,Burkholderia, Enterobacter, Escherichia, Helicobacter, Klebsiella,Mycobacterium, Neisseria, Pseudomonas, Staphylococcus, Streptococcus,Yersinia and the like, including drug resistance strains. In preferredembodiments, the bacteria is B. anthracis (including Ames strain andciprofloxacin resistant Ames strain) B. anth1024, B. brevis, B.lichenifomis, B. megaterium, B. pumilus, B. subtilis, B. vollum, andspores thereof; B. cepacia, B. mallei, M. pseudomallei, and B.thailandensis; E. coli, E. feacalis, E. faecium, and vancomycinresistant strains thereof; K. pneumoniae; P. aeruginosa, preferablyPAO1; S. aureus and methicillin resistant S. aureous; Y. pestis; or acombination thereof.

The activity of the compounds of the present invention may be measuredby any of the methods available to those skilled in the art, includingin vitro and in vivo assays. Examples of suitable assays for activitymeasurements are provided herein. Properties of the compounds of thepresent invention may be assessed, for example, by using one or more ofthe assays set out in the Examples below. Other pharmacological methodsmay also be used to determine the efficacy of the compounds a subjectsuffering from a given disease or disorder. The compounds of the presentinvention may be used in combination with or as a substitution fortreatments known in the art.

The therapeutically effective amounts of the compounds of the inventionfor treating the diseases or disorders described above in a subject canbe determined in a variety of ways known to those of ordinary skill inthe art, e.g. by administering various amounts of a particular compoundto a subject afflicted with a particular condition and then determiningthe effect on the subject. Typically, therapeutically effective amountsof a compound of the present invention can be orally administered dailyat a dosage of the active ingredient of 0.002 to 200 mg/kg of bodyweight. Ordinarily, a dose of 0.01 to 10 mg/kg in divided doses one tofour times a day, or in sustained release formulation will be effectivein obtaining the desired pharmacological effect. It will be understood,however, that the specific dose levels for any particular subject willdepend upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease.

Frequency of dosage may also vary depending on the compound used and theparticular disease treated. It will also be appreciated that theeffective dosage of the compound used for treatment may increase ordecrease over the course of a particular treatment. Changes in dosagemay result and become apparent by standard diagnostic assays known inthe art. In some instances chronic administration may be required. Thecompounds of the present invention may be administered before, during,after, or a combination thereof exposure to bacteria.

As provided herein, an “effective amount” is intended to mean thatamount of a compound that is sufficient to reduce, prevent or inhibitBoNT/A LC metalloprotease activity as compared with a negative control.A “therapeutically effective amount” of a compound of the presentinvention, a prodrug, an active metabolite, or a salt thereof, is aquantity sufficient to, when administered to a subject, reduce, preventor inhibit BoNT/A LC metalloprotease activity. Also, as used herein, a“therapeutically effective amount” of a compound of the presentinvention is an amount which prevents, inhibits, suppresses, or reducesa given clinical condition in a subject as compared to a control. Asdefined herein, a therapeutically effective amount of a compound of thepresent invention may be readily determined by one of ordinary skill byroutine methods known in the art.

The pharmaceutical formulations of the invention comprise at least onecompound of the present invention and may be prepared in a unit-dosageform appropriate for the desired mode of administration. Thepharmaceutical formulations of the present invention may be administeredfor therapy by any suitable route including oral, rectal, nasal, topical(including buccal and sublingual), dermal, mucosal, vaginal andparenteral (including subcutaneous, intramuscular, intravenous andintradermal). It will be appreciated that the preferred route will varywith the condition and age of the recipient, the nature of the conditionto be treated, and the chosen compound of the present invention.

The compound can be administered alone, but will generally beadministered as pharmaceutical formulations suitable for administration.Pharmaceutical formulations known in the art contemplated herein.Pharmaceutical formulations of this invention comprise a therapeuticallyeffective amount of at least one compound of the present invention, andan inert, pharmaceutically or cosmetically acceptable carrier ordiluent. As used herein the language “pharmaceutically acceptablecarrier” or a “cosmetically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical or cosmetic administration. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the formulation is contemplated.Descriptions of suitable pharmaceutically acceptable carriers,formulations, and factors involved in their selection, are found in avariety of readily available sources, e.g., Remington's PharmaceuticalSciences, 17^(th) ed., Mack Publishing Company, Easton, Pa., 1985, whichis incorporated herein by reference.

Supplementary active compounds can also be incorporated into theformulations. Supplementary active compounds include antibiotics,antiprotozoal agents, antifungal agents, and antiproliferative agentsknown in the art, analgesics and other compounds commonly used to treatdiseases and disorders associated with bacterial infection and toxicside effects of bacterial infection including intoxication by a toxin.Supplementary active compounds also include those known in the art whichdelay toxin induced muscle paralysis such as BoNT/A holotoxin inducedmuscle paralysis.

Antibiotics include penicillin, cloxacillin, dicloxacillin, methicillin,nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin,azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin,azithromycin, clarithromycin, clindamycin, erythromycin, lincomycin,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,quinolone, cinoxacin, nalidixic acid, fluoroquinolone, ciprofloxacin,enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin,ofloxacin, sparfloxacin, trovafloxacin, bacitracin, colistin, polymyxinB, sulfonamide, trimethoprim-sulfamethoxazole, co-amoxyclav,cephalothin, cefuroxime, ceftriaxone, vancomycin, gentamicin, amikacin,metronidazole, chloramphenicol, nitrofurantoin, co-trimoxazole,rifampicin, isoniazid, pyrazinamide, kirromycin, thiostrepton,micrococcin, fusidic acid, thiolactomycin, fosmidomycin, and the like.

Antiprotozoal agents include chloroquine, doxycycline, mefloquine,metronidazole, eplornithine, furazolidone, hydroxychloroquine,iodoquinol, pentamidine, mebendazole, piperazine, halofantrine,primaquine, pyrimethamine sulfadoxine, doxycycline, clindamycin, quininesulfate, quinidine gluconate, quinine dihydrochloride,hydroxychloroquine sulfate, proguanil, quinine, clindamycin, atovaquone,azithromycin, suramin, melarsoprol, eflornithine, nifurtimox,amphotericin B, sodium stibogluconate, pentamidine isethionate,trimethoprim-sulfamethoxazole, pyrimethamine, sulfadiazine, and thelike.

Antifungal agents include amphotericin B, fluconazole, itraconazole,ketoconazole, potassium iodide, flucytosine, and the like.

Antiproliferative agents such as altretamine, amifostine, anastrozole,arsenic trioxide, bexarotene, bleomycin, busulfan, capecitabine,carboplatin, carmustine, celecoxib, chlorambucil, cisplatin,cisplatin-epinephrine gel, cladribine, cytarabine liposomal,daunorubicin liposomal, daunorubicin daunomycin, dexrazoxane, docetaxel,doxorubicin, doxorubicin liposomal, epirubicin, estramustine, etoposidephosphate, etoposide VP-16, exemestane, fludarabine, fluorouracil 5-FU,fulvestrant, gemicitabine, gemtuzumab-ozogamicin, goserelin acetate,hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, irinotecan,letrozole, leucovorin, levamisole, liposomal daunorubicin, melphalanL-PAM, mesna, methotrexate, methoxsalen, mitomycin C, mitoxantrone,paclitaxel, pamidronate, pegademase, pentostain, porfimer sodium,streptozocin, talc, tamoxifen, temozolamide, teniposide VM-26,topotecan, toremifene, tretinoin, ATRA, valrubicin, vinorelbine,zoledronate, steroids, and the like.

Supplementary active compounds also include other compounds known in theart which inhibit botulinum neurotoxin serotype A light chainmetalloprotease activity, anthrax lethal factor protease activity, or acombination thereof such as NSC 240898, NSC 266474, NSC 266476, NSC290107, NSC 290108, NSC 290109, NSC 294200, NSC 294201, NSC 294203, NSC294204, NSC 294206, NSC 300511, NSC 308571, NSC 308572, NSC 308574, NSC317880, NSC 317881, NSC 317884, NSC 317885, 317886, NSC 317887, NSC341907, NSC 341909, and NSC 341911.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Various compounds of the present invention have been found to exhibitantibacterial activity and prevent the growth and spore germination ofbacteria such as Bacillus, Burkholderia, Enterobacter, Escherichia,Helicobacter, Klebsiella, Mycobacterium, Neisseria, Pseudomonas,Staphylococcus, Streptococcus, Yersinia and the like, including drugresistance strains. In preferred embodiments, the bacteria is B.anthracis (including Ames strain and ciprofloxacin resistant Amesstrain) B. anth1024, B. brevis, B. lichenifonnis, B. megaterium, B.pumilus, B. subtilis, B. vollum, and spores thereof; B. cepacia, B.mallei, M. pseudomallei, and B. thailandensis; E. coli, E. feacalis, E.faecium, and vancomycin resistant strains thereof; K. pneumoniae; P.aeruginosa, preferably PAO1; S. aureus and methicillin resistant S.aureous; Y. pestis; or a combination thereof. Various compounds of thepresent invention, including NSC 240898, NSC 266474, NSC 266476, NSC290107, NSC 290108, NSC 290109, NSC 294200, NSC 294201, NSC 294203, NSC294204, NSC 294206, NSC 300511, NSC 308571, NSC 308572, NSC 308574, NSC317880, NSC 317881, NSC 317884, NSC 317885, 317886, NSC 317887, NSC341907, NSC 341909, and NSC 341911 are also found to inhibit theprotease activity of anthrax lethal factor.

Thus, not only is the present invention directed to methods ofinhibiting toxin activity, such as botulinum neurotoxin serotype A lightchain metalloprotease activity or the protease activity of anthraxlethal factor, but it is also directed to methods of treating a subjectsuffering from a bacterial infection.

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations, andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

1. A method of inhibiting the activity of Botulinum neurotoxin Ametalloprotease which comprises contacting Botulinum neurotoxin Ametalloprotease with at least one compound having the followingstructural formula:

wherein n is 1 or 2; X¹, X², X³, X⁴, X⁵ and X⁶ are each independently N,S, O, SO₂, CR⁷ or NR⁸ and at least one of X¹ or X² is N, S, O, SO₂, orNR⁸; L is a linker which may be a direct bond or

where Z is an optionally substituted alkyl, alkenyl, dialkenyl,trialkenyl, or aryl, or C(O)NH; and R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ areeach independently hydrogen, amino, amine with stabilized carbocations,carboxyl, optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, alkoxy, aryoxy, cycloalkoxy,heteroaryloxy, alkoxycarbonyl, alkylamino, carbamoyl,alkylaminocarbonyl, alkylsulfhydryl, alkylhydroxymate; and R⁸ ishydrogen, OH, a halogen, or an optionally substituted alkyl.
 2. Themethod of claim 1, wherein at least one of R¹, R², R³, or R⁴ ishydrogen, amidine, 2-imidazoline, amino, guanidine, methyl,aminomethyl-hydroxamine, or methylamine-guanidine.
 3. The method ofclaim 1, wherein R⁵ is hydrogen, amidine, 2-imidazoline, amino,guanidine, methyl, aminomethyl-hydroxamine, methylamine-guanidine,4-oxy-benzamidine, 1H-indole-6-caboxamidine, or1H-indole-5-carboxamidine.
 4. The method of claim 1, wherein R⁶ ishydrogen, amidine, benzamidine, benzimidazoline, imidazoline, guanidine,imidazole, oxazole, benzofuran-2-yl-imidazoline,benzofuran-2-yl-amidine, benzofuran-2-yl-guanidine,benzothiophene-2-yl-imidazoline, benzothiophene-2-yl-amidine,benzene-2-yl-amidine, benzofuran-2-yl-imidazole, orbenzofuran-2-yl-oxazole.
 5. The method of claim 1, wherein at least oneof X¹ or X² is N, NH, S, O, SO₂, CH, C—CH₃, C-phenyl, N-ethanol,N-chloroethyl, C-amino, C-(2-indole-6-imidazoline),C-(2-indole-6-amidine), C-(2-indole-5-imidazoline), orC-(2-indole-5-amidine).
 6. The method of claim 1, wherein at least oneof X³, X⁴, X⁵, or X⁶ is N, NH, S, O, SO₂, or CH.
 7. The method of claim1, wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is —H, —CH₃,—


8. The method of claim 1, wherein R⁵ is —NH₂,


9. The method of claim 1, wherein R⁶ is


10. The method of claim 1, wherein R⁷ is —H, —CH₃, —NH₂,


11. The method of claim 1, wherein R⁸ is —H, —(CH₂)₂OH, or —(CH₂)₂Cl.12. The method of claim 1, wherein L is a direct bond,


13. The method of claim 1, wherein the compound has the followingstructural formulae:


14. A method of inhibiting the activity of Botulinum neurotoxin Ametalloprotease which comprises contacting Botulinum neurotoxin Ametalloprotease with at least one compound selected from the groupconsisting of NSC 92833, NSC 103699, NSC 103701, NSC 130681, NSC 240890,NSC 240891, NSC 240893, NSC 240894, NSC 240895, NSC 240896, NSC 240897,NSC 240898, NSC 240899, NSC 240900, NSC 266472, NSC 266474, NSC 266475,NSC 266476, NSC 266477, NSC 266482, NSC 278995, NSC 278996, NSC 278997,NSC 278999, NSC 290107, NSC 290108, NSC 290109, NSC 290111, NSC 291103,NSC 294199, NSC 294200, NSC 294201, NSC 294202, NSC 294203, NSC 294204,NSC 294206, NSC 294207, NSC 294208, NSC 294494, NSC 300509, NSC 300510,NSC 300511, NSC 300512, NSC 302569, NSC 308569, NSC 308570, NSC 308571,NSC 308572, NSC 308573, NSC 308574, NSC 317880, NSC 317881, NSC 317883,NSC 317884, NSC 317885, NSC 317886, NSC 317887, NSC 328398, NSC 330687,NSC 330688, NSC 330689, NSC 330690, NSC 341082, NSC 341907, NSC 341909,NSC 341910, NSC 341911, NSC 352341, NSC 369718, NSC 369721, NSC 607617,and NSC
 12155. 15. The method of claim 14, wherein the compound is NSC341909, NSC 308574, NSC 240898, NSC 341907, NSC 266472, NSC 330690, NSC278999, NSC 308571, NSC 290107, NSC 290108, NSC 294200, NSC 317884, NSC317884, NSC 294203, NSC 294494, NSC 317881, NSC 330688, NSC 317886, NSC317833, NSC 328398 NSC 352341, NSC 294204, NSC 341911, NSC 300511, NSC607617, NSC 294202, NSC 317880, NSC 240899, NSC 294201, NSC 291103, NSC308573, NSC 290109, NSC 294206, NSC 308570, NSC 294199, NSC 369723, orNSC
 300510. 16. A method of treating, inhibiting or preventing a subjectfrom being intoxicated by Botulinum toxin which comprises administeringto the subject a therapeutically effective amount of at least onecompound having the following structural formula:

wherein n is 1 or 2; X¹, X², X³, X⁴, X⁵ and X⁶ are each independently N,S, O, SO₂, CR⁷ or NR⁸ and at least one of X¹ or X² is N, S, O, SO₂, orNR⁸; L is a linker which may be a direct bond

or where Z is an optionally substituted alkyl, alkenyl, dialkenyl,trialkenyl, or aryl, or C(O)NH; and R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ areeach independently hydrogen, amino, amine with stabilized carbocations,carboxyl, optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, alkoxy, aryoxy, cycloalkoxy,heteroaryloxy, alkoxycarbonyl, alkylamino, carbamoyl,alkylaminocarbonyl, alkylsulfhydryl, alkylhydroxymate; and R⁸ ishydrogen, OH, a halogen, or an optionally substituted alkyl.
 17. Acompound having the following structural formula:

wherein R^(a) and R^(b) are each independently —CN, —CONH₂, or—C(═NH)NH₂; X is —NH or O; and Y is N or —CH,


18. A method of inhibiting the activity of Botulinum neurotoxin Ametalloprotease which comprises contacting Botulinum neurotoxin Ametalloprotease with at least one compound of claim
 17. 19. A method oftreating, inhibiting or preventing a subject from being intoxicated byBotulinum toxin which comprises administering to the subject atherapeutically effective amount of at least one compound of claim 17.20. A pharmaceutical composition comprising at least one compound ofclaim 17 and a pharmaceutically acceptable carrier.